3-methanesulfonylpropionitrile for treating inflammation and/or pain

ABSTRACT

The present invention relates to purified 3-methanesulfonylpropionitrile or a pharmaceutically acceptable salt thereof, and a method for preparing such compound. The compound has at least 90% (w/w) purity. The present invention is also directed to a pharmaceutical composition comprises the purified compound and a pharmaceutically acceptable carrier. The present invention is further directed to a method for treating inflammation, inflammatory-related disorders, or pain, by administering 3-methanesulfonylpropionitrile or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.

This application is a continuation of U.S. application Ser. No.15/349,161, filed Nov. 11, 2016; which is a continuation of U.S.application Ser. No. 14/639,781, filed Mar. 5, 2015, now U.S. Pat. No.9,492,420; which is a continuation of U.S. application Ser. No.14/216,664, filed Mar. 17, 2014, now U.S. Pat. No. 8,975,297; which is acontinuation of U.S. application Ser. No. 13/759,676, filed Feb. 5,2013, now U.S. Pat. No. 8,829,046; which is a divisional application ofU.S. application Ser. No. 13/324,777, filed Dec. 13, 2011, now U.S. Pat.No. 8,476,316; which claims the benefit of U.S. Provisional ApplicationNo. 61/423,485, filed Dec. 15, 2010. The contents of theabove-identified applications are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition comprisinga pharmaceutically acceptable carrier and a compound of3-methanesulfonylpropionitrile, or its pharmaceutically acceptablesalts. The compound is purified to ≥90% purity. The present inventionalso relates to processes of preparing the compound and methods of usingthe purified compound for treating inflammation or inflammatory-relateddisorders and pain.

BACKGROUND OF THE INVENTION

Inflammation is a process by which microbes or tissue injury induce therelease of cytokines and chemokines from various cell types producingincreased blood vessel permeability, upregulation of endothelialreceptors, and thus increased egress of various cells of the innate andadaptive immune system which enter surrounding tissue and grosslyproduce the classical picture of inflammation, i.e. redness, swelling,heat and pain.

Inflammation is a localized reaction of live tissue due to an injury,which may be caused by various endogenous and exogenous factors. Theexogenous factors include physical, chemical, and biological factors.The endogenous factors include inflammatory mediators, antigens, andantibodies. Endogenous factors often develop under the influence of anexogenous damage. An inflammatory reaction is often followed by analtered structure and penetrability of the cellular membrane. Endogenousfactors, namely, mediators, antigens, and autogens define the nature andtype of an inflammatory reaction, especially its course in the zone ofinjury. In the case where tissue damage is limited to the creation ofmediators, an acute form of inflammation develops. If immunologicreactions are also involved in the process, through the interaction ofantigens, antibodies, and autoantigens, a long-term inflammatory processwill develop. Various exogenous agents, for example, infection, injury,radiation, also provide the course of inflammatory process on amolecular level by damaging cellular membranes which initiatebiochemical reactions.

Connective tissues are subjected to a constant barrage of stress andinjury. Acute or chronic impacts and the natural progression of variousdegenerative diseases all produce painful inflammation in joint regions,such as the neck, back, arms, hips, ankles and feet. These afflictionsare common and often debilitating.

Current therapies are directed to some or all of the pathogeneticcomponents of inflammation. For example, corticosteroids have a broadspectrum of activities and NSAIDS are more specificallyanti-prostaglandin and analgesic. All current therapies have relativelyhigh rates of adverse effects and adverse effects are severe andserious.

There is a need for a composition and a method for treatinginflammation, inflammatory-related disorders, and pain. The compositionshould be economic and easy to manufacture, and the method should beeffective and have no significant side effects.

SUMMARY OF THE INVENTION

The present invention is also directed to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a purified compoundof 3-methanesulfonylpropionitrile or a pharmaceutically acceptable saltor solvate thereof. The compound is purified to at least 90% purity(w/w).

The present invention is also directed to a method for treatinginflammation, inflammatory-related disorders, and pain. The methodcomprises the step of administering 3-methanesulfonylpropionitrile or apharmaceutically acceptable salt thereof to a subject in need thereof.The pharmaceutical composition comprising the active compound can beapplied by any accepted mode of administration including topical, oral,and parenteral (such as intravenous, intramuscular, subcutaneous orrectal). Topical administration and oral administration are preferred.

Purified 3-methanesulfonylpropionitrile can be prepared by a methodcomprising the steps of: (a) mixing methionine, a water-immiscibleorganic solvent, and a halogenating agent, and reacting at a temperaturebetween 0-35° C.; (b) removing the aqueous phase and obtaining anorganic solvent phase; and (c) removing the organic solvent to obtainthe compound in an oil form or in a solid form.

Purified 3-methanesulfonylpropionitrile can also be prepared by a methodcomprising the steps of: (a) mixing methionine, ethyl acetate, and anaqueous hypochlorite solution and reacting at a temperature between0-35° C., (b) removing the aqueous phase and obtaining an ethyl acetatephase, (c) reducing the ethyl acetate phase volume to 1-20% of itsoriginal volume by distillation, vacuum, or nitrogen purging, (d) addingethanol to the ethyl acetate of (c), (e) removing the ethyl acetate ofstep (d) and obtaining the compound in the ethanol, (f) reducing thevolume of the ethanol of (e) to cause the compound to precipitate and/orcrystallize out of the ethanol as a solid, and (g) isolating the solid.This method is suitable for large scale and results in high purity (atleast 99%) of the compound.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the total ion spectra (HPLC-TIC) of the product of Example1.

FIG. 2 shows the mass spectra analysis results (HPLC-MS) of the productof Example 1.

DETAILED DESCRIPTION OF THE INVENTION Definition

An “adduct”, as used herein, is a product of a direct addition of two ormore distinct molecules, resulting in a single reaction productcontaining all atoms of all components. An “adduct ion” is formed from aprecursor ion and contains all of the constituent atoms of that ion aswell as additional atoms or molecules. Adduct ions are often formed in amass spectrometer ion source.

“Pharmaceutically acceptable salts,” as used herein, are salts thatretain the desired biological activity of the parent compound and do notimpart undesired toxicological effects. Pharmaceutically acceptable saltforms include various crystalline polymorphs as well as the amorphousform of the different salts. The pharmaceutically acceptable salts canbe formed with metal or organic counterions and include, but are notlimited to, alkali metal salts such as sodium or potassium; alkalineearth metal salts such as magnesium or calcium; and ammonium ortetraalkyl ammonium salts, i.e., NX₄+ (wherein X is C₁₋₄).

“Solvates,” as used herein, are addition complexes in which the compoundis combined with an acceptable co-solvent in some fixed proportion.Co-solvents include, but are not limited to, ethyl acetate, lauryllactate, myristyl lactate, cetyl lactate, isopropyl myristate, methanol,ethanol, 1-propanol, isopropanol, 1-butanol, isobutanol, tert-butanol,acetone, methyl ethyl ketone, acetonitrile, benzene, toulene, xylene(s),ethylene glycol, dichloromethane, 1,2-dichloroethane, N-methylformamide,N,N-dimethylformamide, N-methylacetamide, pyridine, dioxane, and diethylether.

Purified Compound

The present invention is directed to a purified compound of3-methanesulfonylpropionitrile:

The present invention is also directed to the pharmaceuticallyacceptable salts, or solvates of 3-methanesulfonylpropionitrile.

The compound preferably has a purity of at least 85%, 90%, 95%, 97%,98%, or 99%.

The inventor has discovered that 3-methanesulfonylpropionitrile can beprepared by a method comprising the steps of: (a) mixing methionine, awater-immiscible organic solvent, and a halogenating agent, and reactingat a temperature between 0-35° C.; (b) removing the aqueous phase andobtaining an organic solvent phase; and (c) removing the organic solventto obtain the compound in an oil form or in a solid form.

Methionine can be L-methionine, D-methionine, or a mixture thereof.

Halogenating agents useful for this invention include fluorinatingagents, chlorinating agents, and brominating agents. Examples ofhalogenating agents are hypochlorite, chloramine T, chlorine gas,hydrogen bromide, phosphorus tribromide, phosphorus pentabromide, and1-chloromethyl-4-fluoro-1, 4-diazoniabicyclo[2.2.2]octanebis-(tetrafluoroborate). A preferred chlorinating agent is hypochlorite(e.g., sodium hypochlorite). Commercial bleach CLOROX® contains about 6%hypochlorite, and can be used as a halogenating agent.

The water-immiscible organic solvent useful in this invention ispreferably a semi-polar or non-polar solvent having a polarity of about0.1-7.5, such as ethyl acetate, hexane, heptane, methylene chloride,n-butanol, lauryl lactate, myristyl lactate, cetyl lactate, or isopropylmyristate. A preferred water-immiscible organic solvent is ethylacetate.

The reaction of step (a) is carried out at a temperature between 0° C.to ambient temperature, for example 0-35° C., preferably 4-30° C., andmore preferably 22-28° C. The reaction is preferably carried out underbasic conditions, for example, between pH 7.1-14, preferably, pH 7.2-9,or pH 7.5-8.

In one embodiment, methionine is in a solid form and is mixed with awater-immiscible organic solvent and an aqueous halogenating agent. Themixing is optionally carried out under an inert gas, e.g. argon.

In a preferred embodiment, solid methionine is mixed with awater-immiscible organic solvent first and an aqueous halogenating agentis then added to the rapidly stirred suspension. The organic solvent isin excess of water by at least about 2-fold, 5-fold, or 10-fold involume. “About” as used in this application, refers to ±15% of therecited value. For example, the volume of the organic solvent is about2-20 fold, 3-10 fold, or 4-6 fold of the water volume. The reaction ofmethionine and hypochlorite is very fast in water and is difficult tocontrol. The excess organic solvent slows down the reaction, and thusthe reaction is more controlled and reproducible. The reaction time isat least 1 hour, 2 hours, 4 hours, 8 hours, or 12 hours, depending onthe scale of the production. The reaction time is typically 2-24, or4-16, or 8-16 hours. The reaction is often carried overnight forconvenience.

In a less preferred embodiment, an aqueous solution of a halogenatingagent is added to methionine (either in a solid form or an aqueoussolution form) and thoroughly mixed, and then the organic solvent isadded immediately,

After the methionine/halogenating agent reaction occurs, the reactiveproduct partitions in the organic solvent.

The mixing of step (a) can be done by any means of mechanical mixing,for example, impeller stirrer, sheer mixing, rotary mixing, etc.

After the reaction of step (a) is complete, the water-organic solventmixture is allowed to settle. The organic phase is separated from thewater phase by any means known to a skilled person such as decanting orpipetting, and the organic solvent extract containing the reactiveproduct is obtained. Preferably, the organic phase is collected in thepresence of sodium sulfate to remove residual water. Any non-solubleresidues in the organic solvent extract and sodium sulfate areoptionally removed by filtration, decanting, centrifugation, or anymeans known to a skilled person. The reactive product is stable (withoutsignificant oxidation or hydrolysis) in the organic solvent at roomtemperature (22-28° C.) for at least a month, preferably, 3 months, morepreferably 6 months or a year.

In a typical reaction, 10-200 g of methionine, and 200 mL-4 L of 3-12%(e.g. 6%) hypochlorite are used. In a typical extraction, about 1-20 Lor more water-immiscible organic solvent is used. The amounts of theabove reagents can be scaled up or scaled down proportionally.

In a preferred embodiment, the water-immiscible organic solvent is ethylacetate. After the aqueous phase is removed, the ethyl acetate solventis removed from the product by any means known to a person skilled inthe art. For example, the ethyl acetate solvent can be removed byevaporation such as rotary evaporation or drying under nitrogen gas.After the ethyl acetate solvent is removed, the product3-methanesulfonylpropionitrile is obtained in an oil form or in a solidform, which is stored in an enclosed vessel (such as a capped vial orbottle) and is stable for at least 1-3 months at room temperature(22-28° C.).

After the ethyl acetate solvent is removed, the solid product isidentified as 3-methanesulfonylpropionitrile by infusion massspectroscopy, HPLC-MS, Time of Flight High resolution MS, elementalanalysis, ¹H NMR, ¹³C NMR, and FTIR.

After the ethyl acetate solvent is removed, the oil or solid product3-methanesulfonylpropionitrile in general has at least 80% (w/w) purity.The product can be further purified to remove impurities by preparativechromatography such as thin-layer chromatography, column chromatography,and HPLC, recrystallization, solvent washing, or other suitable means.Preferred purification methods include thin-layer chromatography andflash chromatography. After purification, 3-methanesulfonylpropionitrilehas purity of at least about 85%, or 90%, or 93%, or 95%, or 98%, or99%. The synthetic yield of the purification procedure is in general15-40%.

In another embodiment of the invention, 3-methanesulfonylpropionitrilecan be prepared by a process comprising the steps of (a) mixingmethionine, ethyl acetate, and an aqueous hypochlorite solution andreacting at a temperature between 0-35° C., (b) removing the aqueousphase and obtaining an ethyl acetate phase, (c) reducing the ethylacetate phase volume to 1-20% (e.g. 5-10%) of its original volume bydistillation, vacuum, or nitrogen purging, (d) adding ethanol to theethyl acetate of (c), (e) removing the ethyl acetate of step (d) andobtaining the compound in the ethanol, (f) reducing the volume of theethanol of (e) to cause the compound to precipitate and/or crystallizeout of the ethanol solution as a solid, and (g) isolating the solid. Thesolid is either amorphous powder or in a crystalline form.

In the above process, after step (c) and before step (d), the ethylacetate solution is optionally dried by a suitable means to remove anyresidual water, e.g., drying over magnesium sulfate. In the step (d),the volume of ethanol added is the same or in excess of the volume ofethyl acetate. For example, the volume of ethanol can be 1-10 fold(preferably 1-5 fold or 1-3 fold) of that of the ethyl acetate. Ethanolis added to the ethyl acetate phase and azeotropic displacement of ethylacetate is conducted under vacuum or nitrogen purge. The volume ofethanol is reduced by vacuum, nitrogen purge, or distillation (step(e)). After the solid is precipitate and/or crystallize out of theethanol solution as a solid, it can be isolated by vacuum filtration.The isolated solid can be dried by suitable means such as ambient airdrying or vacuum drying. The solid can be further purified byre-crystallization, re-slurry/precipitation, and/or column purification,to obtain purity of greater than 98 or 99%.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprisingone or more pharmaceutically acceptable carriers and3-methanesulfonylpropionitrile, or a pharmaceutically acceptable salt,or solvate thereof. The active compound 3-methanesulfonylpropionitrile,or its pharmaceutically acceptable salt or solvate in the pharmaceuticalcompositions in general is in an amount of about 0.01-20%, or 0.05-20%,or 0.1-20%, or 0.2-15%, or 0.5-10%, or 1-5% (w/w) for a topicalformulation; about 0.1-5% for an injectable formulation, 0.1-5% for apatch formulation, about 1-90% for a tablet formulation, and 1-100% fora capsule formulation.

In one embodiment, the active compound is incorporated into anyacceptable carrier, including creams, gels, lotions or other types ofsuspensions that can stabilize the active compound and deliver it to theaffected area by topical applications. In another embodiment, thepharmaceutical composition can be in the dosage forms such as tablets,capsules, granules, fine granules, powders, syrups, suppositories,injectable solutions, patches, or the like. The above pharmaceuticalcomposition can be prepared by conventional methods.

Pharmaceutically acceptable carriers, which are inactive ingredients,can be selected by those skilled in the art using conventional criteria.Pharmaceutically acceptable carriers include, but are not limited to,non-aqueous based solutions, suspensions, emulsions, microemulsions,micellar solutions, gels, and ointments. The pharmaceutically acceptablecarriers may also contain ingredients that include, but are not limitedto, saline and aqueous electrolyte solutions; ionic and nonionic osmoticagents such as sodium chloride, potassium chloride, glycerol, anddextrose; pH adjusters and buffers such as salts of hydroxide,phosphate, citrate, acetate, borate; and trolamine; antioxidants such assalts, acids and/or bases of bisulfite, sulfite, metabisulfite,thiosulfite, ascorbic acid, acetyl cysteine, cystein, glutathione,butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, andascorbyl palmitate; surfactants such as lecithin, phospholipids,including but not limited to phosphatidylcholine,phosphatidylethanolamine and phosphatidyl inositiol; poloxamers andploxamines, polysorbates such as polysorbate 80, polysorbate 60, andpolysorbate 20, polyethers such as polyethylene glycols andpolypropylene glycols; polyvinyls such as polyvinyl alcohol andpovidone; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose andhydroxypropyl methylcellulose and their salts; petroleum derivativessuch as mineral oil and white petrolatum; fats such as lanolin, peanutoil, palm oil, soybean oil; mono-, di-, and triglycerides; polymers ofacrylic acid such as carboxypolymethylene gel, and hydrophobicallymodified cross-linked acrylate copolymer; polysaccharides such asdextrans and glycosaminoglycans such as sodium hyaluronate. Suchpharmaceutically acceptable carriers may be preserved against bacterialcontamination using well-known preservatives, these include, but are notlimited to, benzalkonium chloride, ethylene diamine tetra-acetic acidand its salts, benzethonium chloride, chlorhexidine, chlorobutanol,methylparaben, thimerosal, and phenylethyl alcohol, or may be formulatedas a non-preserved formulation for either single or multiple use.

For example, a tablet formulation or a capsule formulation of3-methanesulfonylpropionitrile may contain other excipients that have nobioactivity and no reaction with the active compound. Excipients of atablet may include fillers, binders, lubricants and glidants,disintegrators, wetting agents, and release rate modifiers. Binderspromote the adhesion of particles of the formulation and are importantfor a tablet formulation. Examples of binders include, but not limitedto, carboxymethylcellulose, cellulose, ethylcellulose,hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch,starch, and tragacanth gum, poly(acrylic acid), andpolyvinylpyrrolidone.

For example, a patch formulation of 3-methanesulfonylpropionitrile maycomprise some inactive ingredients such as 1,3-butylene glycol,dihydroxyaluminum aminoacetate, disodium edetate, D-sorbitol, gelatin,kaolin, methylparaben, polysorbate 80, povidone, propylene glycol,propylparaben, sodium carboxymethylcellulose, sodium polyacrylate,tartaric acid, titanium dioxide, and purified water. A patch formulationmay also contain skin permeability enhancer such as lactate esters(e.g., lauryl lactate) or diethylene glycol monoethylether.

Topical formulations including 3-methanesulfonylpropionitrile can be ina form of gel, cream, lotion, liquid, emulsion, ointment, spray,solution, and suspension. The inactive ingredients in the topicalformulations for example include, but not limited to, lauryl lactate(emollient/permeation enhancer), diethylene glycol monoethylether(emollient/permeation enhancer), DMSO (solubility enhancer), siliconeelastomer (rheology/texture modifier), caprylic/capric triglyceride,(emollient), octisalate, (emollient/UV filter), silicone fluid(emollient/diluent), squalene (emollient), sunflower oil (emollient),and silicone dioxide (thickening agent).

In one embodiment, lauryl lactate (for example, at about 0.1-10%, orabout 0.2-5%, or about 0.5-5%) is included in the topical gelformulation. Lauryl lactate is considered safe for topicaladministration. Lauryl lactate is qualified for human use withinpharmaceutical and cosmetic products. Lauryl lactate when used in atopical formulation enhances the permeability of the compound.Preferably lauryl lactate is purified to achieve ≥90%, preferably ≥95%purity; the high purity mitigates the presence of hydrolytic andoxidative agents. In addition, DMSO at 0.1-20%, or 0.5-10% (w/w) in theformulation provides suitable solubility of3-methanesulfonylpropionitrile.

In another embodiment, diethylene glycol monoethylether is included inthe topical gel formulation.

Method of Use

Inflammation is a process and a state of tissue pathology resulting fromactivation and continuation of activity of the innate and acquiredcomponents of the immune system. The arachidonic acid cascade andcytokine production and action in cell to cell interactions are criticalcomponents of immune activation and response, which lead toinflammation. Arachidonic acid resides in many cell membranes. Whenarachidonic acids are cleaved from the membranes, it can produce many ofthe known eicosinoids including prostaglandins and leucotrienes, whichare known pro-inflammatory entities.

Applicant has discovered that 3-methanesulfonylpropionitrile inhibitedpro-inflammatory cytokine release (e.g., IL-1β, IL-6, TNFα, IL-4 andIFNγ) from human peripheral blood mononuclear cells in vitro. Applicanthas discovered that 3-methanesulfonylpropionitrile is anti-inflammatorywhen applied topically in the mouse ear swelling model, in which theinflammation was induced by arachidonic acid. Applicant has found that agel formulation containing 3-methanesulfonylpropionitrile was welltolerated in 14-day dermal toxicity studies in rats and minipigs. Theonly effects seen after oral, systemic toxicity administration in ratsand dogs were mild physiological effects including decreased bodytemperature, decreased respiratory rate, increased blood pressure andincreased heart rate. The effects were seen at doses over 2000-foldabove expected human therapeutic doses, which indicates that thecompound would be well tolerated for systemic therapeutic use.

The present invention is directed to a method of treating inflammationand/or pain. The active compound 3-methanesulfonylpropionitrile can beused as is, or it can be administered in the form of a pharmaceuticalcomposition that additionally contains a pharmaceutically acceptablecarrier. The method comprise the steps of first identifying a subjectsuffering from inflammation and/or pain, and administering to thesubject 3-methanesulfonylpropionitrile, in an amount effective to treatinflammation and/or pain. “An effective amount,” as used herein, is theamount effective to treat a disease by ameliorating the pathologicalcondition or reducing the symptoms of the disease.

In one embodiment, the method reduces or alleviates the symptomsassociated with inflammation. The present invention provides a method totreat localized manifestations of inflammation characterized by acute orchronic swelling, pain, redness, increased temperature, or loss offunction in some cases.

In another embodiment, the present invention provides a method toalleviate the symptoms of pain regardless of the cause of the pain. Thegeneral term “pain” treatable by the present method includes traumaticpain, neuropathic pain, organ pain, and pain associated with diseases.Traumatic pain includes pain resulting from injury, post-surgical painand inflammatory pain. Neuropathic pain includes neuropathic andidiopathic pain syndromes, and pain associated with neuropathy such asdiabetic neuropathy, causalgia, brachial plexus avulsion, occipitalneuralgia, fibromyalgia, gout, and other forms of neuralgia. Organ painincludes ocular, corneal, bone, heart, skin/burn, visceral (kidney, gallbladder, etc.), joint, and muscle pain. Pain associated with diseasesincludes pain associated with cancer, AIDS, arthritis, herpes andmigraine. The present invention reduces pain of varying severity, i.e.mild, moderate and severe pain; acute and chronic pain. The presentinvention is effective in treating joint pain, muscle pain, tendon pain,and burn pain.

In preferred embodiments, the present invention is useful in treatinginflammation and/or pain associated in a musculoskeletal system or onthe skin. The highly innervated, musculoskeletal and skin systems have ahigh capacity for demonstration of pain. In addition, themusculoskeletal system has a high capacity for tissue swelling, and theskin has a high capacity for redness, swelling, and heat. Inmusculoskeletal and skin systems, the degree of tissue damage isfrequently magnified out of proportion to the resulting inflammatoryresponse. In the skin for example, merely firm stroking will causerelease of the cytokines, IL-1 and TNF.

The present invention provides a method for treating inflammation and/orpain associated with inflammatory skeletal or muscular diseases orconditions. The method comprises the steps of identifying a subject inneed thereof, and administering to the subject the active compound, inan amount effective to treat inflammation and/or pain. The skeletal ormuscular diseases or conditions include musculoskeletal sprains,musculoskeletal strains, tendonopathy, peripheral radiculopathy,rheumatoid arthritis, polymyalgia rheumatica, juvenile arthritis, gout,ankylosing spondylitis, psoriatic arthritis, systemic lupuserythematosus, costochondritis, tendonitis, bursitis, such as the commonlateral epicondylitis (tennis elbow), medial epichondylitis (pitcherselbow) and trochanteric bursitis, temporomandibular joint syndrome, andfibromyalgia.

The present invention provides a method for treating inflammation and/orpain associated with inflammatory skin diseases such as dermatitis andpsoriasis. The method comprises the steps of identifying a subject inneed thereof, and administering to the subject the active compound, inan amount effective to treat inflammation and/or pain.

Skin is highly reactive to environmental stimuli and the epidermalcomponent of keratinocytes is a very rich source of both arachidonicacid and pro-inflammatory cytokines of IL-1 and TNF. The skin dendriticcells, Langerhans cells, recognize and process antigens for furtherimmune response of various lymphocytes and all of these cells areprimarily regulated by cytokines through their specific cell surfacereceptors.

Dermatitis (also called eczema) is generic inflammation of the skin.Specific types of dermatitis include atopic, contact, nummular, andphoto-induced.

Contact dermatitis has two types, the non-specific irritant type and theantigen specific allergic type. Both involve innate and acquired immunesystem response including arachidonic acid and cytokine components thatinitiate and propagate the disease through cell to cell messaging byeicosanoid and/or cytokine moieties produced by epidermal cells,macrophages, dendritic cells, neutrophils, eosinophils, and various Tand B lymphocytes.

Atopic dermatitis is eventually a specifically Th2 lymphocyte mediateddisease. The initiator of atopic dermatitis is stimuli of epidermalkeratinocytes, arachidonic acid and cytokine release. It is establishedthat eicosanoid inhibitors such as prostaglandin inhibitors and cytokineinhibitors such as cyclosporine, tacrolimus, and various specificcytokine receptor monoclonal antibodies reduce the symptoms of atopicdermatitis.

Psoriasis is currently believed to be initiated by some type of injuryor microbial stimulus to the epidermis, which causes arachidonic acidand cytokine release and initiates and propagates an inappropriateimmune response, which causes the disease. Inhibition of these changesby various eicosanoid and cytokine inhibitors such as prostaglandininhibitors and cyclosporine and the newer monoclonal antibodies directedagainst specific cell surface receptors for cytokines has beendemonstrated to improve the disease.

3-methanesulfonylpropionitrile, which is effective in inhibitingarachidonic acid induced inflammation and in inhibiting the release ofpro-inflammatory cytokine, is effective to treat inflammation and/orpain associated with psoriasis and dermatitis, particularly contactdermatitis, and atopic dermatitis.

The pharmaceutical composition of the present invention can be appliedby local administration and systemic administration. Localadministration includes topical administration. Systemic administrationincludes oral, parenteral (such as intravenous, intramuscular,subcutaneous or rectal), and other systemic routes of administration. Insystemic administration, the active compound first reaches plasma andthen distributes into target tissues. Topical administration and oraladministration are preferred routes of administration for the presentinvention.

Dosing of the composition can vary based on the extent of the injury andeach patient's individual response. For systemic administration, plasmaconcentrations of active compounds delivered can vary; but are generally1×10⁻¹⁰-1×10⁻⁴ moles/liter, and preferably 1×10⁻⁸-1×10⁻⁵ moles/liter.

In one embodiment, the composition is applied topically onto theaffected area and rubbed into it. The composition is topically appliedat least one or two times a day, or 3 to 4 times per day, depending onthe medical issue and the disease pathology being chronic or acute. Ingeneral, the topical composition comprises about 0.01-20%, or 0.05-20%,or 0.1-20%, or 0.2-15%, 0.5-10, or 1-5% (w/w) of the active compound.For example, the topical composition comprises about 1 or 5% (w/w) ofthe active compound. Depending on the size of the affected area, 0.2-85mL, typically 0.2-10 mL, of the topical composition is applied to theindividual per dose. The active compound passes through skin and isdelivered to the site of discomfort.

In one embodiment, the pharmaceutical composition is administratedorally to the subject. The dosage for oral administration is generally1-50, and preferably 1-5 mg/kg/day.

In one embodiment, the pharmaceutical composition is administratedsubcutaneously to the subject. The dosage for subcutaneousadministration is generally 0.3-20, and preferably 0.3-3 mg/kg/day.

Those of skill in the art will recognize that a wide variety of deliverymechanisms are also suitable for the present invention.

The present invention is useful in treating a mammal subject, such ashumans, horses, and dogs. The present invention is particularly usefulin treating humans.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

EXAMPLES Example 1. Preparation of Product (Starting Material 15 g)

DL-Methionine (approximately 15 g, 100 mmol) was weighed into a 2 Lreaction flask. 1.5 L of reagent grade ethyl acetate was added to theflask and to the rapidly stirred via mechanical means. To the suspensionwas added 310 mL of CLOROX® bleach (about 6% sodium hypochlorite). Theflask was capped and stirring was continued at room temperature forabout 18 hours.

The mixture was transferred to a separatory funnel, the lower aqueousphase was drained off. The organic layer was dried over sodium sulfateto remove water, and then filtered and washed with 225 mL of ethylacetate.

The organic phase was added to a 2 L Erlenmeyer flask and concentratedby nitrogen purge to about 230 mL volume. It was transferred to a to a0.5 L Erlenmeyer flask with 5 mL rinse of ethyl acetate and wasconcentrated further by nitrogen purge to an off white powder andsubmitted for analysis. This material was confirmed to be3-methanesulfonylpropionitrile sodium adduct with 97% purity (seeExample 3).

Example 2. Preparation of Product (Starting Material 109 g)

DL-Methionine (approximately 109 g, 716 mmol) was weighed into a 20 Lreactor containing 10.9 L of reagent grade ethyl acetate and to therapidly stirred suspension was added 2200 mL (2420 g) of CLOROX® bleach(1951 mmol). The reactor was capped and stirring was continued at roomtemperature for a period of 18 hrs.

Stirring was halted and the mixture was allowed to settle for 30minutes. The lower aqueous layer was drained off. The organic layer wasdried over magnesium sulfate, and filtered and washed with anapproximate additional 2 L of ethyl acetate, which resulted inapproximately 13 L of organic phase.

Incrementally the organic phase was added to 6 L rotary evaporator at 30mbar, bath temperature of 22° C. and condenser temperature of −4° C.,until it was concentrated to about 5% of the initial volume(approximately 0.65 L). An equal volume of ethanol (Absolute, 200 Proof,≥99.5%, ACS Reagent) was added and azeotropic displacement of ethylacetate was conducted by maintaining the pot volume (about 1.3 L) withincremental additions of ethanol, which resulted in the precipitation ofa white solid in ethanol. The resulting slurry was filtered, washed withethanol, and dried; the solid was collected as 18.3 g of 92% purematerial (Crop 1). The filtrates were refrigerated overnight, whichresulted in additional solid precipitate being formed; the solidprecipitate was collected by vacuum filtration, washed with ethanol, anddried to yield 4.2 g (Crop 2) with a purity of 96%.

The first and second crops were combined in a 500 mL round bottom flaskwith 250 mL ethanol, heated to 55-60° C. and slowly cooled to 5±3° C.After completion of a two hour age, the product slurry was filtered,washed with cold ethanol, and dried by vacuum, which yielded 21.3 g of99.7% pure material in a solid form.

The purity and identification of the material(3-methanesulfonylpropionitrile sodium adduct) were confirmed by theHPLC-MS method described in Example 3.

Example 3. Identification of Product as 3-Methanesulfonylpropionitrile

The isolated product of Example 1 was analyzed by infusion massspectroscopy, HPLC-MS, Pos mode, Time of Flight High resolution MS,Elemental analysis, ¹H and ¹³C NMR and FTIR.

1. HPLC-MS

HPLC-MS Instrument Parameters are listed in Table 1 below.

TABLE 1 Instrument: Agilent model 1100 w/MSD Mobile Phase: 85% Methanol,13% Ethanol, 2% IPA and 0.1% TFA Flow: 0.3 mL/min Stop Time: 17 minColumn Temp: 30° C. Injection Volume: 0.5 μL Source: ElectrosprayPolarity: Positive Gas Temp: 350° C. Drying Gas Flow: 13.0 L/minNebulizer Press: 60 psig Scan Parameters Mass Range: 50-500 AMUFragmentor Voltage: 50 V

Total ion spectra were collected from 75 to 500 AMU (Atomic mass unit).The Total Ion Chromatogram (FIG. 1) demonstrates that the test samplehas purity of 96.76% (at 12.3 minute) based on total area under curve.FIG. 2 is the mass spectrum of the 12.3 minute peak from FIG. 1. FIG. 2depicts the intensity of the signal versus the mass to charge ratio(directly relevant to the molecular weight). FIG. 2 depicts the majorisolated ion as 156.1 AMU and a minor ion as 134.1, which is consistentwith the product being 3-methanesulfonylpropionitrile with a molecularweight (M) of 133.1, assayed as the M+1 (M+proton, 134.1) and M+23(M+Na, 156.1, sodium adduct) by mass spectroscopy.

2. TOF High Resolution MS

TOF High Resolution MS Instrument Parameters are listed in Table 2.

TABLE 2 Instrument: Waters ACQUITY UHPLC-QTof micro mass spectrometer &LockSpray accurate mass inlet installed Mobile Phase: 85% Methanol, 13%Ethanol, 2% IPA and 0.1% TFA Flow: 0.3 mL/min Stop Time: 20 min ColumnTemp: 30° C. Injection Volume: 1.0 μ/L Source: Electrospray Polarity:Positive Capillary voltage: 2500 V Sample Cone 15.0 V voltage:Extraction Cone 1.5 V voltage: Desolvation Temp: 300° C. Source Temp:110° C. Cone gas flow: 10 L/hr Desolvation gas 600 L/hr flow:The time of flight (TOF) high resolution HPLC-MS chromatogram peak at12.3 minutes contains an ion at 156.0078 m/z in its spectrum. The ionobserved at 156.0078 m/z in the spectrum of the chromatographic peak at12.3 minutes is likely a sodium adduct. The elemental composition of thesodium adduct is C₄H₇NO₂S+Na with an error of 10.9 ppm. The highresolution TOF data supports the HPLC ES-MS data that the test sample ofExample 1 is 3-methanesulfonylpropionitrile with a molecular weight 133AMU.3. Elemental Analysis

The elemental analysis data resulted in elemental composition of 36.385%C, 5.560% H, 10.304% N, 25.14% S provides supporting data of theelemental composition of the compound as C₄H₇NO₂S.

4. 1H NMR

1H NMR Instrument Parameters are listed in Table 3.

TABLE 3 Solvent CD₃OD Transmitter Nucleus Proton Spectrometer Frequency(sfrq) 399.798 (MHz) Temp (° C.) 25 D1 (sec) 5 Spin (Hz) 20 Processing(1D) LB (Hz) 0.2 # points acquired 35k Pw (us) 6 Processing size (fn)64k Sweep width (sw) (kHz) 7 Number of Transients (nt) 40

The 1H NMR spectrum was acquired in CD₃OD solution at 400 MHz bySpectral Data Services, Inc. The 1H NMR spectrum comprises threediscrete and integratable signals, as shown in Table 4.

TABLE 4 ¹H Chemical shifts (ppm) and coupling constants (J in Hz) inCD₃OD. Chemical Number of Shift (ppm) RIV^(a) Protons Multiplicity J(Hz) Assignment 2.989 20.00 2 t 7.05 CH ₂ 3.056 28.79 3 s CH ₃ 3.48819.37 2 t 7.05 CH ₂ ^(a)Relative Integration Value

There is also the characteristic CD₃OD multiplet centered at 3.307 ppmand minor impurity peaks at: ˜1.1 ppm (RIV=0.11); ˜2.1 ppm (RIV=0.16);˜4.2 ppm (RIV=0.08); ˜4.6 ppm (RIV=0.02); 4.79 ppm (RIV=0.08).

The 1H NMR spectrum is indicative of a general structure shown belowthat includes: (a) adjacent methylene groups, each of which is attachedto a unique electron withdrawing group/heteroatom (b) an isolated methylgroup, which is attached to an electron withdrawing group/heteroatom:

-   -   X and Y are electron withdrawing groups/heteroatoms        5. C¹³ NMR

C¹³ NMR Parameters are listed below in Table 5.

TABLE 5 Solvent CD₃OD Transmitter Nucleus Carbon Spectrometer Frequency(sfrq) 100.590 (MHz) Temp (° C.) 25 D1 (sec) 5 Spin (Hz) 0 Processing(1D) LB (Hz) 3 # points acquired 17664 Pw (us) 15 Processing size (fn)32k Sweep width (sw) (kHz) 25.2 Number of Transients (nt) 500 DecouplerNuclei (dn) H Decoupler Frequency 399.797 Dmm www Dm yyy

The 13C NMR spectrum was acquired in CD₃OD solution at 100 MHz bySpectral Data Services, Inc. The 13C NMR spectrum comprises fourdiscrete, integratable signals, as shown in Table 6.

TABLE 6 ¹³C Chemical shift data. Chemical shift (ppm) ArbitraryAssignment 118.641 CN 50.285 C(alkyl)X 41.029 C(alkyl)Y 12.068 C(alkyl)Z

The 13C NMR spectrum indicates four unique types of carbon atoms. Thedata is corroborative of the 1H NMR data, only if X or Y in structure 1is carbon. The line at 118 ppm is characteristic of a nitrile group,i.e., Y═CN. The remaining two signals are suggestive of carbons attachedto a heteroatom other than oxygen and, since the compound resulted fromchemistry where methionine was the starting material, it is reasonablethat atom X is sulfur, giving rise to the following structure:

Considering the chemistry employed (treatment of methionine with excesssodium hypochlorite in the form of aqueous bleach), S-oxidation would beunavoidable, giving rise to structures the following structures:

The mass spectrum acquired displayed a prominent ion at m/z 156, and alesser ion at m/z 134. The latter ion is consistent with the parent ionof structure C acquired in the positive mode, i.e., M+1, whereas theformer ion is likely a sodium ion adduct (M+23). Further support forstructure C has been provided by high-resolution mass spectroscopy,which gave an exact mass of 156.0078, which is consistent with amolecular formula of C4H7NaNO2S. Therefore, the proton and carbon 13 NMRspectra are supportive of the structure of3-methanesulfonylpropionitrile.6. Fourier Transform Infrared Spectrometer (FTIR)

Fourier Transform Infrared Spectrometer (FTIR) Instrument Parameters arelisted below in Table 7.

TABLE 7 Spectral reflectance Probe Yes Scan range (cm-1)  650-4000 Mode% T Mode range (%) 26.8-100The FTIR data depicts a spectra consistent with the presence of thenitrile functionality with a N≡C stretch at 2255.7 cm-1 and strong bandsat 1129.5 and 1276.5 consistent with the presence of

The resultant spectrum is supportive of the structure of3-methanesulfonylpropionitrile.

Example 4. Gel Formulation 1

Table 8 exemplifies one gel formulation containing3-methanesulfonylpropionitrile.

TABLE 8 5% Gel 1% Gel 3-methanesulfonylpropionitrile   5.0%   1.0% DowCorning Elastomer Blend EL-8050 ID  61.0%  69.0% Labrafac Lipophile WL1349  8.0%  8.0% Octisalate  5.0%  5.0% Lauryl Lactate  1.1%  3.2%Dimethyl Sulfoxide (DMSO)  8.9%  1.8% Dow Corning 556 Cosmetic GradeFluid  7.0%  7.9% Squalene  2.0%  2.0% Sunflower Seed Oil  2.0%  2.0%Dow Corning Aerogel VM-2270  0.1%  0.0% 100.0% 100.0%

Example 5. Gel Formulation 2

Table 9 exemplifies another gel formulation containing3-methanesulfonylpropionitrile.

TABLE 9 5% Gel 3-methanesulfonylpropionitrile   5.0% Diethylene GlycolMonoethylether   5.0% Acrylates/C10-30 alkyl acrylate crosspolymer(CARBOPOL ® Ultrez 20  0.50% polymer) Trolamine(tris(2-hydroxyethyl)amine)  0.47% Purified Water  89.03%  100.0%

Example 6. Anti-Inflammatory and Analgesic Activity of3-Methanesulfonylpropionitrile (Prophetic Example)

Purified 3-methanesulfonylpropionitrile (MSPN, prepared according toExample 2) is prepared in the gel formulation according to Example 5.Test materials: MSPN in gel formulation (1-5%), indomethacin (positivecontrol), and vehicle (gel formulation without active compound) areevaluated for anti-inflammatory and analgesic activity in the ratcarrageenan-induced paw inflammation model.

Rats are used in the experiment. Carrageenan (0.1 mL of a 1% suspension)is injected subcutaneously into the left hind paw to induceinflammation. MSPN (1-5%) or vehicle gel is applied to the paw topicallyat volumes of 0.05, 0.1 0.15 or 2.0 mL, 1.5, 2.5, and 3.5 hoursfollowing the carrageenan administration. Indomethacin is given orallyat 5 mg/kg, 1 hour prior to carrageenan administration. The degree ofinflammation (edema, or swelling) is determined using a plethysmographto measure paw volume. Analgesia is determined by measuring pawwithdrawal to a mechanical stimulus using von Frey filaments.Inflammation and analgesia are measured 4 hours after carrageenanadministration. MSPN is expected to have anti-inflammatory and/oranalgesic properties as measured by a significant decrease in paw volumeand/or a significant increase in mechanical pressure needed to elicitpaw withdrawal, respectively, as compared to the vehicle control(t-test, p<0.05).

Example 7. Analgesic Activity of 3-Methanesulfonylpropionitrile(Prophetic Example)

Purified 3-methanesulfonylpropionitrile (MSPN, prepared according toExample 2) is prepared in the gel formulation according to Example 5.Test materials: active compound in gel formulation (1-5%), morphine(positive control), and vehicle (gel formulation without activecompound) are evaluated for analgesic activity in the rat hot platemodel.

Rats are used in the experiment. MSPN (1-5%) or vehicle gel is appliedto the paw topically at volumes of 0.05, 0.1 0.15 or 2.0 mL. One hourlater the rat is placed on a 55° C. hot plate, and the time to lick thepaw is measured. The positive control, morphine, is given orally at 30mg/kg, 1 hour prior to hot plate testing. MSPN are expected to haveanalgesic properties as measured by a significant increase in time tolicking as compared to the vehicle control (t-test, p<0.05).

Example 8. Treatment of Knee Pain (Prophetic Example)

Objectives:

To investigate the efficacy of 3-methanesulfonylpropionitrile in a gelformulation in patients with mild to moderate knee pain associated withosteoarthritis following temporary cessation of standard NSAID therapy.

Formulation:

The gel formulation contains 3-methanesulfonylpropionitrile at 1% and 5%(Example 5) are used in this example. Placebo contains the same gelwithout the active compound.

Methodology:

A randomized, double-blind, placebo controlled, parallel treatmentmulticenter Phase 2 clinical activity study.

Patients with painful osteoarthritis of the knee, controlled by a stabledose of standard NSAID therapy for at least 2 months, discontinue use ofthe NSAIDs for a 7 day washout period. Patients are then randomized in a1:1:1 ratio (1% active gel, 5% active gel, placebo). A total of up to150 patients are enrolled and treated for 7 days with follow-up at 8,10, 14 and 21 days.

The active Gel or placebo is applied to the affected knee 3 times a dayfor 7 days for a total of 21 treatments given every 4-6 hours whileawake.

Patients are treated for 7 days and followed up for a further 14 days.NSAIDs may be restarted after the Day 10 visit.

Criteria for Evaluation:

Safety:

-   -   Adverse Events (AEs) throughout the study.    -   Physical examination at enrollment (−7 days, start of NSAID        washout period), Baseline (Day 1, start of treatment), Day 10        and Day 21.    -   Vital signs at enrollment (−7 days, start of NSAID washout        period), Baseline (Day 1, start of treatment) and Days 2, 4, 8,        10, 14 and 21.    -   Clinical laboratory measurements at Baseline (Day 1), Day 8 and        Day 14.

Clinical Activity:

The primary clinical activity parameters are the measurement of pain atthe site of application, as quantified by VAS and the Western Ontarioand McMaster University (WOMAC) scale. The effect of treatment onswelling, tenderness and inflammation of the knee is recorded, also thetime to reduction or eradication of pain after treatment is recorded.

Study Endpoints:

The primary clinical activity endpoint is:

-   -   Change from Baseline (Day 1) to Day 8 in WOMAC functional        disability index:    -   Pain (Scale 0-20).    -   Stiffness (Scale 0-8).    -   Physical function (Scale 0-68).

The secondary clinical activity endpoints are:

-   -   Change from Baseline (Day 1) to Day 8 in VAS pain scale (1-100).    -   Within-day change in VAS pain scale on Day 2 and Day 3 as        measured by change from daily Baseline (Pre-Treatment 1) to 30        minutes Post Treatment 2.    -   Change in investigator evaluation of swelling, tenderness and        inflammation between Baseline (Day 1) and 30 minutes and 60        minutes after the first application on Day 1.    -   Change in investigator evaluation of swelling, tenderness and        inflammation between Baseline (Day 1) and Day 8.    -   Time to reduction or eradication of pain subsequent to each        topical application of active gel or placebo gel.    -   Use of rescue medication (APAP).

Example 9. Inhibition of Cytokine Activities

3-methanesulfonylpropionitrile (MSPN, prepared according to Example 1was tested for its effects on in vitro cytokine release from humanperipheral blood mononuclear cells (PBMCs). Secretion of cytokines byPBMCs plays a significant role in the inflammatory response.

MSPN was added to cultures of fresh human PBMCs at 162 μM (22 μg/mL) induplicate. One hour later, PBMCs were stimulated to secrete cytokinesusing the mitogens lipopolysaccharide and concanavalin A (ConA).Lipopolysaccharide at 50 μg/mL was used to stimulate the release ofinterleukin IL-1β, IL-6 and tumor necrosis factor TNFα. ConA at 20 μg/mLwas used to stimulate the release of IL-4 and ConA at 5 μg/mL was usedto stimulate interferon IFNγ. The corticosteroid dexamethasone (100 nM)was used as a positive control. After 24 hours of incubation, thesupernatants were assayed for the cytokines using the Luminex Bead kit.MSPN at 22 μg/mL inhibited the release of IL-1β, IL-6, TNFα, IL-4 andIFNγ by 95%, 98%, 98%, 7% and 21%, respectively. Dexamethasone inhibitedthe release of IL-1β, IL-6, TNFα, IL-4 and IFNγ by 24%, 60%, 42%, 93%and 87%, respectively.

The results demonstrate that MSPN has a significant inhibitory effect oncytokines involved in the inflammatory process.

Example 10. Anti-Inflammatory Activity of 3-Methanesulfonylpropionitrilein Mice

Purified 3-methanesulfonylpropionitrile (MSPN, prepared according toExample 2) was dissolved in vehicle (ethanol/acetone 1:1) to 5% (w/v).The active compound, indomethacin (positive control in vehicle), andvehicle were evaluated for anti-inflammatory activity in the topicalarachidonic acid induced ear swelling model in mice.

Male ICR derived mice weighing 22±2 g were used in this experiment. 5mice were used for each group (active compound, positive control, andvehicle). All animals were maintained in a controlled temperature(22-24° C.) and humidity (60%-70%) environment with 12-hour light/darkcycles for at least one week prior to use.

Arachidonic acid (2 mg in 20 μL acetone) was applied topically onto theanterior and posterior surfaces of the right ear of test animals toinduce inflammation. MSPN in vehicle (20 μL), indomethacin (0.3 mg) invehicle, and vehicle (20 μL) was each applied 30 minutes before and 15minutes after arachidonic acid challenge. At 90 minutes afterarachidonic acid induction of ear edema, the thickness of the right earand the left ear was measured and the difference calculated as anindication of the inflammation in the right ear. Significant activity isdefined as a reduction (inhibition) in arachidonic acid induced earswelling by ≥30% relative to the vehicle-treated group.

MSPN and indomethacin both caused a significant decrease (31 and 60%,respectively) in the ear swelling induced by arachidonic acid at 90minutes, relative to the vehicle-treated group (acetone:ethanol/1:1).The difference between MSPN-treated mice and vehicle-treated mice wasalso determined to be statistically significant (p-value determined byt-test was <0.05).

Example 11. Systemic Administration of MSPN Formulation

This study was done to determine the systemic (plasma) exposure of MSPNafter administration by the oral and subcutaneous routes to rats.

MSPN substance was prepared in water for oral administration and insaline for subcutaneous administration. Rats weighed 282 to 295 g wereused in the study. Male rats (n=2) were given a single dose at 50, 160or 500 mg/kg by both oral and subcutaneous routes. Female rats (n=2)were dosed only at 500 mg/kg by both oral and subcutaneous routes. Theblood was drawn from each rat at 0.25, 1, 2, 3, 4, 6, 12, 24, and 48hours and measured for MSPN concentration by LC/MS/MS.

For males, the average maximum plasma concentrations measured (Cmax)after oral dosing at 50, 160 or 500 mg/kg were 160, 560 and 12,000μg/mL, respectively; and after subcutaneous dosing were 160, 760 and3300 μg/mL, respectively. For females, the average Cmax after oraldosing of 500 mg/kg was 3800 μg/mL, and after subcutaneous dosing of 500mg/kg was 9500 μg/mL. Half-lives were similar by both routes and forboth sexes, and ranged from 8 to 15 hours.

The above results demonstrate that there was significant bioavailabilityof MSPN after both the oral and subcutaneous routes.

Example 12. Treating Pain and/or Inflammation Emanating from Ligaments,Tendons, Muscles, Joints, Bones, and Fascia in Humans

Materials:

Each patient was provided with a gel formulation containing 5% of3-methanesulfonylpropionitrile (see Example 5).

Treatment of Knee

Patient A is 73-year old male whose right knee has a long history ofcompromise and 3 surgeries. Following 2 days of skiing, patient's rightknee was swollen and sore. There was particular discomfort in the tendonthat runs along the outside right of the knee. There was also pronouncedtenderness underneath the bottom portion of the kneecap. Circumferenceof the knee was 16⅞ inches.

Patient A applied the 5% gel formulation on the right knees twice 7hours apart. Three hours after the second application, there was nonoticeable swelling on the knee and no pain under kneecap. There wasonly slight discomfort remaining on outside ligament. The circumferenceof knee after treatment was 16⅜ inches, which is a reduction of ½inches.

Treatment of Finger

Patient A also has a long history of arthritis in fingers of the lefthand, especially in the middle finger. Patient A felt noticeableswelling, discomfort, and troubling stiffness in the middle finger andwas unable to bend the finger beyond 45°. Circumference of knuckle ofthe middle finger was 3 5/16 inches.

Patient A applied the 5% gel formulation on the middle finger once.About 9 hours after treatment, patient noticed that the stiffness of thefinger was relieved and there was no remaining discomfort. Patient A wasable to fully bend the middle finger to make a closed fist.Circumference of knuckle of the middle finger after treatment was 215/16 inches, which is a reduction of ⅜ inches.

Treatment of Neck

Patient B is a female at age 74, who 15 years ago had both a nervestimulator implanted, and had a nerve decompression surgery in her neck.For 15 years, Patient B was pain free. In 2011, the same pain from 15years ago returned. Patient B applied one treatment of 5% gelformulation. After rubbing it in well, she then applied a heating padfor about one hour on the neck. Patient B recorded that pain anddiscomfort was relieved for several hours, prior to the nextapplication. Patient B repeated this treatment 3 times per day for 2days and each time recorded that her pain was relieved.

Patient C is an 88-year old male. Patient C pulled muscles in the backof his lower neck after swinging a 6-pound indoor training golf club.Three hours later the pain was excruciating. Patient C applied the 5%gel formulation; at that time Patient C recorded that the pain was a 10on a scale of 1 to 10 (10 being the worst). The pain remained intenseduring the next fours hours; Patient C recorded that during this timethe pain only dropped to an 8 on the scale. Then the second applicationof the 5% gel formulation was made and by the end of the next 6 hours,Patient C said the pain had dropped down to a 4 on the scale. Then thethird application of 5% gel formulation was made. Four hours later,Patient C recorded that the pain dropped down to a 1 on the scale andstayed there for next six hours, at which time the fourth and finalapplication was made. Within the next six hours, all pain waseliminated.

Patient D is a 59-year old male. After approximately a 30-mile bikeride, Patient D aggravated an arthritic condition in his neck andaggravated strained muscles in the back of his lower neck. After rubbingin only one application the 5% gel formulation, Patient D felt majorrelief of pain and stiffness within one hour.

Treatment of Shoulder

Patient E is a 58-year old male. Patient E developed and experiencedleft shoulder pain below the rotator cuff and into the bicep for sevendays from playing golf. Patient E continued to play golf and his injurywas not healing. Patient E applied the 5% gel formulation twice a dayfor 3 subsequent days while continuing to play golf. Patient E feltimmediate reduction of pain after each application of the 5% gelformulation, and all pain and inflammation was eliminated after 3 days.

Treatment of Hand & Fingers

Patient F is a 74-year old male. Patient F pulled tendons in thumb whenplaying golf. Patient F felt excruciating soreness and saw visibleswelling on thumb. Immediately after the golf game, Patient F appliedthe 5% gel formulation to the afflicted area of his thumb. Within fourhours, the pain had dramatically reduced and the range of motion of thethumb had improved. Four hours later before bed, Patient F appliedanother treatment and the pain and swelling continued to be reduced. Bythe next morning, pain was entirely eliminated and swelling waseliminated.

Patient G is a 72-year old man who had chronic arthritis flare-ups inhis fingers. Patient G had a flare-up of arthritis in his right thumb.There was no noticeable swelling, but there was a strong aching painthat was pervasive to the entire thumb. The pain was so bad that hecould not use his right hand. Patient G applied one application of the5% gel formulation all over his entire right thumb and massaged it infor about 5 minutes. After six hours, all pain was eliminated.

Treatment of Wrist

Patient F also suffers from chronic pain and inflammation on the wrist,also referred to as gout-like symptoms. When Patient F felt pain andinflammation on the wrist, he applied the 5% gel formulation on wristthree times a day every six hours for 2 days. Patient F felt relief onehour after the first application and by the end of the 2 days, pain anddiscomfort was removed. Patient F has done this treatment several times.

Treatment of Ankle

Patient F further suffers from a chronic condition of achillestendonitis in his right ankle. When his ankle flared up, it usually tooka ‘cycle’ of 10 days to two weeks to return back to normal. When thepatient applied 5% gel formulation on ankle three times a day for 2days, the recovery ‘cycle’ time to normal is reduced by half the numberof days.

Treatment of Chest

Patient H is a 55-year old female who has had a chronic condition ofcostochondritis. When Patient H had flare-up, she experienced sharpstabbing-like pains and tenderness in the area of her sternum. The paindebilitated her for days. With the onset of pain, Patient H applied the5% gel formulation on afflicted area every 6 hours for two to four days.Within 2 hours of first application, pain was substantially reduced.After 24 hours of treatment (3 applications), Patient H debilitatingfeeling and soreness to touch was eliminated. Dull pain remained throughthe second day of application. With repeated applications, dull pain waseliminated and Patient H stopped treatment.

Treatment of Fascia

Patient I is an 85-year old female who has a chronic condition ofplantar fasciitis in her right foot. When she suffered an onset of pain,Patient I applied the 5% gel formulation on the bottom of her foot andmassaged it in for about 5 minutes. Patient I typically did thistreatment at night before bed, and once during the day when she stayedoff her foot for several hours. Three hours after treatment, patientfelt initial reduction of pain. Within 24 hours of treatment, pain waseliminated.

The invention, and the manner and process of making and using it, arenow described in such full, clear, concise and exact terms as to enableany person skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims. To particularly point out and distinctly claim thesubject matter regarded as invention, the following claims conclude thespecification.

What is claimed is:
 1. A method of reducing or alleviating symptoms oflocalized manifestation of inflammation, comprising the steps of:identifying a subject suffering from localized manifestation ofinflammation, and administering to the subject a compound of3-methanesulfonylpropionitrile, or a pharmaceutically acceptable solvatethereof, in an amount effective to treat the localized manifestations ofinflammation, wherein localized manifestation of inflammation ischaracterized by acute or chronic swelling, pain, redness, or increasedtemperature.
 2. The method according to claim 1, wherein said localizedmanifestation of inflammation is characterized by redness.
 3. The methodaccording to claim 1, wherein said localized manifestation ofinflammation is characterized by increased temperature.
 4. The methodaccording to claim 1, wherein said compound is orally administered. 5.The method according to claim 1, wherein said compound is topicallyadministered.
 6. A pharmaceutical composition comprising apharmaceutically acceptable carrier and 3-methanesulfonylpropionitrileor a pharmaceutically acceptable solvate thereof, wherein thecomposition is in an oral form of a tablet, and the pharmaceuticallyacceptable carrier is selected from the group consisting of:carboxymethylcellulose, cellulose, ethylcellulose,hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch,starch and tragacanth gum, poly(acrylic acid), and polyvinylpyrrolidone.